Cheng Xiaoshu

Plasma exosomes induced by remote ischaemic preconditioning attenuate myocardial ischaemia/reperfusion injury by transferring miR-24

Remote ischaemic preconditioning (RIPC) is well known to protect the myocardium against ischaemia/reperfusion injury (IRI). Exosomes are small extracellular vesicles that have become the key mediators of intercellular communication. Various studies have confirmed that circulating exosomes mediate RIPC. However, the underlying mechanisms for RIPC-induced exosome-mediated cardioprotection remain elusive. In our study, we found that the expression level of miR-24 was higher in exosomes derived from the plasma of rats subjected to RIPC than in exosomes derived from the plasma of control rats in vivo. The rat plasma exosomes could be taken up by H9c2 cells. In addition, miR-24 was present in RIPC-induced exosomes and played a role in reducing oxidative stress-mediated injury and decreasing apoptosis by downregulating Bim expression in H2O2-treated H9c2 cells in vitro. In vivo, miR-24 in RIPC-induced exosomes reduced cardiomyocyte apoptosis, attenuated the infarct size and improved heart function. Furthermore, the apoptosis-reducing effect of miR-24 was counteracted by miR-24 antagomirs or inhibitors both in vitro and in vivo. Therefore, we provided evidence that RIPC-induced exosomes could reduce apoptosis by transferring miR-24 in a paracrine manner and that miR-24 in the exosomes plays a central role in mediating the protective effects of RIPC.

GW24-e0353 A novel mutation R397Q in KCNQ1 gene caused dilated cardiomyopathy with ventricular tachycardia and prolonged QT interval

Objectives Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death with lethal ventricular arrhythmias. So far, the genetic mutations of DCM are mainly related to the sarcomeric protein, and mutations in SCN5A which encoding μ-subunit of sodium channel also have been involved. However, KCNQ1 gene encoding in potassium channel has not been found in DCM patients. The purpose of this study was to demonstrate if DCM with ventricular tachycardia and prolonged QT interval is caused by mutation in the KCNQ1 gene. Methods Clinical features and DNA samples of DCM patients with lethal ventricular arrhythmia were collected. The candidate genes including SCN5A, KCNQ1, KCNE1 and KCNE2 were screened by the direct sequencing to look for the mutations. Whole cell patch clamp analysis of HEK293 cells expressing Wild-type and mutation KCNQ1 channel was used to investigate the channels biophysical properties. The current-voltage relationships, peak current, current density, the voltage dependence of steady-state activation and inactivation of mutated channel were investigated. Results Twelve unrelated DCM patients with lethal ventricular arrhythmia were included and clinical features were available in medical records. Three patients (25%) presented cardiac conduction system abnormality, and nine patients (75%) showed QT prolongation in ECG recordings before amiodarone therapy. A G to A missense mutation in KCNQ1 gene was identified in a 60-year-old man with concomitant DCM and incessant VT and prolonged QT interval, which is at nucleotide site 1190 that resulted in a amino-acid substitution of arginine to glutamine at amino-acid site 397 (R397Q) in the C-terminal region of KCNQ1 gene. Whole cell patch clamp analysis of HEK293 cells expressing WT and mutant KCNQ1 was used to investigate the biophysical properties of the channel. It showed that R397Q (n = 19) decreased Iks current density dramatically compared with WT (14.34 ± 1.73 vs 23.25 ± 2.31, P < 0.01). However, R397Q didn’t influence on the V0.5 and slope factor for the voltage dependence of activation comparing to WT. Meanwhile, this patient experienced recurrent VT only 20 months post radio frequency ablation (RFCA). Finally an implantable cardioverter defibrillator (ICD) was successfully implanted and he had been without complaints up to the 3 years of follow-up visit. Conclusions It was firstly reported that a loss of function in KCNQ1 gene may cause the overlapping phenotype of DCM, ventricular tachycardia and prolonged QT interval. The reduction of Iks in R397Q did not significantly change the properties of gate. It seems that RFCA is the assistant to improve the incessant ventricular arrhythmia in the patients with abnormal structure heart disease and genetic background, ICD is necessary.

MicroRNA-223 prevents cardiomyocyte hypertrophy by targeting cardiac troponin I-interacting kinase

Objectives Our study was designed to investigate the role of microRNA-223 (miR-223) and its direct target gene, cardiac troponin I-interacting kinase (TNNI3K), in regulating cardiomyocyte hypertrophy. Methodology Neonatal rat cardiomyocytes (CMs) were cultured from one to two days old Sprague–Dawley rats. Cardiomyocyte hypertrophy was induced by endothelin-1 (ET-1). Expression of miR-223 in CMs was detected by real-time PCR. miR-223 mimics transfection was performed to achieve overexpression of miR-223 in CMs. Cell size was measured via surface area calculation under fluorescence microscopy after anti-α-actinin staining. Expression levels of ANP, α-actinin, Myh6, Myh7, as cardiac hypertrophy related marker genes, were detected by RT-PCR. The expression of TNNI3K protein was analysed by western blot. Luciferase assay was performed to confirm the direct binding of miR-223 to the 3′UTR of TNNI3K mRNA. Results In ET-1 induced hypertrophic CMs, expression of miR-223 was lower than that in normal CMs (Normal CMs: 1.00±0.08 vs Hypertrophic CMs: 0.62±0.16, p<0.05). Under stimulation of ET-1, miR-223 overexpressed CMs showed alleviated hypertrophic phenomenon, which characterised by less cell surface area (miR-223 group: 2590±781 mm2 vs ET-1 only group: 4680±1040 mm2, p<0.01) and lower expression of ANP, α-actinin, Myh6, Myh7, when compared with ET-1 stimulation only CMs. In miR-223 overexpressed CMs, the expression of TNNI3K protein was significantly decreased (miR-223 group: 0.39±0.05 vs Control: 0.03±0.01). Co-transfection of a miR-223 expression vector with pMIR-TNNI3K led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that TNNI3K is a direct target gene of miR-223. Conclusion All these results suggest that TNNI3K, a novel cardiac-specific kinase gene, is a direct target of miR-223. miR-223 plays a important role as suppressor in cardiomyocyte hypertrophy and could be used in clinical treatment of hypertrophy in future.

GW25-e1598 Transcatheter occlusion of ventricular septal defect after Fallot's tetralogy repair

To report experiences of transcatheter occlusion of ventricular septum residual leakage after Fallot s tetralogy repair. Chose 9 inpatients (average age was 15.68±5.20 year old) who were diagnosed with ventricular septum residual leakage after Fallot’s tetralogy repair according to history and

GW24-e2990 The expression of rock in cardiomyocte exposed to hypoxia and its role in cardiomyocte injury

Objectives Using cultured myocardial cells in vitro and establishing virtual hypoxia environment of cardiomyocyte apoptosis model, to observe the expression of Rock in myocyte exposed to different hypoxia phase and its role in the mechanism of apoptosis. Methods 1. Cardiomyocytes from the hearts of 1-3-day-old neonatal rats were prepared. 2. Established myocardial cell apoptosis model, prepared the simulated hypoxia liquid which have been deoxygenated by bubbling with the mixture of 95% nitrogen and 5% carbon dioxcide for 1 hours. Then exposed to it for 1 h,3 h,6 h,9 h, respectively. Select the time when Rock-1and Rock-2 expression the highest, put the PI3K/AKT blocking agent and Erk5 blocking agent into hypoxia liquid respectively, and then detect related indicators. 3. The cell apoptosis and viability were detected by flow cytometry and MTS respectively. The expression of Rock-1, Rock-2, p-Erk5, Eek5, Caspase-3, PI3k, p-Pi3k, GSK3β at different hypoxia phase were detected by western blotting. Results 1. After exposed to hypoxia liquid for 1 h, 3 h, 6 h, 9 h respectively, the rate of myocyte apoptosis increased and survival rates decreased. After 1 h, Rock-1, Rock-2 and Erk5 in hypoxia-ischaemia liquid began to rise, reached its peak in 3-6 hours, in 9 hours began to decrease, which were significantly higher than the control group (P < 0.01); Caspase-3, Gsk3β in hypoxia-ischaemia liquid, began to rise, which was sustained expression in a high state at following observed time. There was no significace of the expression of Pi3k in the course of hypoxia-ischaemia which was no statistical significance. But After 1 h, p-Pi3k in hypoxia-ischaemia liquid began to rise, reached its peak in 6 hours, in 9 hours began to decrease, which were significantly statistical significance between the two groups (P < 0.05); 3. Put PI3K/AKT blocking agent LY294002 (final concentration 20 umol / L) into liquid, pretreatment for 2h, and then, cardiomyocytes were treated in hypoxia liquid for 6h, myocardial cell apoptosis compared with the group of hypoxia 6 hours was significantly higher (P < 0.01), the survival rate compared with hypoxia group decreased significantly (P < 0.01). The expression of Caspase-3, Rock-1 and Rock-2 were also significantly higher compared with hypoxia 6 h group (P < 0.05) 4. Put Erk5 blocking agent LY294002 (final concentration 20 umol/L) into liquid, pretreatment for 2 h, and then, cardiomyocytes were treated in hypoxia liquid for 6 h, myocardial cell apoptosis compared with the group of hypoxia 6 hours was significantly higher (P < 0.01), the survival rate compared with hypoxia group decreased significantly (P < 0.01). The expression of Rock-1 and Rock-2 were also significantly higher compared with hypoxia 6h group. Conclusions 1. Rock-1 and Rock-2 are involved in the process of hypoxia-induced cardiomyocyte apoptosis, which may play an important role by inhibiting the PI3K/AKT pathways. 2. Erk5 perform anti-apoptosis effection through reducing the activation of Rocks. 3. Hypoxia can activate Gsk3β, Caspase-3, thereby increasing cardiac myocyte apoptosis. Hypoxia can promote cardiac myocyte apoptosis through activating Gsk3β and Caspase-3 expression.

GW24-e2379 Expression of prohibitin and proliferation in vascular smooth muscle cells after balloon injure in rat carotid artery

Objectives To study the expression Prohibitin in proliferation vascular smooth muscle cells (VSMCs) after balloon injure in rat carotid artery. Methods Twenty-four male SD rats of SPF grade were randomly divided into control groups with 6 animals and injured group with 18 animals. Based on the model of balloon injure in rat carotid artery, the artery tissues were harvested on 7, 14, 28 days after operation. The following investigation was carried out: Immunohistostaining and Western-blotting analysis the expression of Prohibitin in proliferation VSMCs. Results The arterial intimal thickening and neointima formation, at 7 days after vascular balloon catheter injury, there were 4-6 layers of cells. 14 days after balloon injury, there were more than 8 ∼ 10 layers of cells, and injury induces arterial intimal thickening after 28 days. The intimal proliferation area was more thickener in 28 days than 14 days, and 14 days more thickener than 7 d after injur, the 7 days more thickener than the control group, P < 0.05. There was PHB expressed in VSMCs of normal vascular, but PHB expression was significantly increased in injured rat carotid arteries on 7 day compared with normal and further to increase on 14 day and 28 day after injury (P < 0.05) Conclusions The results indicated there was a small amount PHB expressed in VSMCs of normal vascular, the expression of PHB may be associated with VSMCs proliferation and migration after balloon injury.

GW24-e2991 Protective effect of cardiotrophin-1 in preventing acute cardiomyocyte hypoxia- reoxygeonation injury mediated by supressor of cytokine signalling 1--SOCS1/stat3 pathway

Objectives To investigate the signalling pathways supressor of cytokine signalling 1 (SOCS1) how to involve in the protective effect of CT-1 in preventing cardiomyocyte I/R injury from cellular and molecular aspects. Methods Cardiomyocytes from the hearts of 1∼3-day-old neonatal rats (Sprague-Dawley) were prepared by a modification of a previously published protocol. There were seven groups in the experiment: 1) control group: cultivated with DMEM for 6h; 2) hypoxia-reoxygeonation group: 3) CT-1 group; 4) ASODN group1 and group 2: antisense oligonucleotides group (two antisense SOCS1 groups) : 5) SODN group (sense oligonucleotides SOCS1 group); 6) ScODN group (scrambled oligonucleotides SOCS1 group). Myocytes survival rate was evaluated by MTS method, apoptosis, mitochondrial permeability transition pore (Δψm) and reactive oxygen species (ROS) were detected by flow cytometer. SOCS1 and STAT3 protein by western blotting. Results Cardiomyocyte apoptosis and ROS increased markedly after hypoxia/reoxygenation, but cardiomyocyte survival rate and the level of Δψm decreased significantly. With CT-1 intervention, cardiomyocyte survival rate increased markedly (87%), apoptosis and ROS reduced significantly Δψm was lower and the ROS was higher than those in CT-1 group after giving antisense SOCS1 infection, but it was similar to that in CT-1 group after sense SOCS1 or scrambled SOCS1 infection. After hypoxia-reoxygeonation, Expression of SOCS1 mRNA and SOCS1 protein increased somewhat comparing with control group, but there no significant difference. However,expression of both SOCS1 mRNA and SOCS1 protein in CT-1 group increased apparently comparing with hypoxia-reoxygeonation group. Phosphorylated STAT3 increased obviously after processing with SOCS1 antisense oligonucleotides. Conclusions Supressor of cytokine signalling 1--SOCS1/STAT3 pathway may mediate the protection of CT-1 in preventing acute cardiomyocyte hypoxia- reoxygeonation injury.

GW24-e3848 The organ protective effect of direct renin inhibitor in a mouse model of type 2 diabetes

Objectives The benefit of blocking the renin-angiotensin system (RAS) with conventional RAS blockers in cardiovascular diseases and nephropathy of patients with type 2 diabetes is now well established. Direct renin inhibitor (DRI) as a novel renin-angiotensin system inhibitor has been used in hypertensive patients. However, the efficacy of DRI in type 2 diabetes has not been fully established. In this study, we firstly investigated the protective effect of aliskiren in a mouse model of type 2 diabetes. Methods Groups of db/db mice (C57BLKS/J-leprdb/leprdb), a mouse model of obesity and type 2 diabetes, were treated with aliskiren (3, 6, 12 and 25 mg/kg/day) or hydralazine (80 mg/kg/day) for 6 weeks, and non-diabetic db/m mice (C57BLKS/J-leprdb/+) were used as control. The protective effects were extensively compared among groups. Results Except the lowest dose of aliskiren group, the other doses of aliskiren significantly decreased the blood pressure (BP) of db/db mice. All sub-pressor and hypotensive doses of aliskiren significantly attenuated cardiac fibrosis, cardiac macrophage infiltration, coronary remodelling, albuminuria, renal glomerulus sclerosis, renal macrophage infiltration and improved vascular endothelial function in db/db mice. These protective effects of aliskiren were attributed to the attenuation of cardiac p22phox-related NADPH oxidase-induced superoxide and the restoration of vascular endothelial nitric oxide synthase production. Aliskiren at the highest dose partially reduced glucose intolerance in db/db mice. Furthermore, the highest dose significantly attenuated the decreases in pancreatic islet insulin content and beta cell mass, and prevented pancreatic islet fibrosis, being associated with the reduction of 8-hydroxy-2’-deoxyguanosine-positive cells and Nox2 expression in pancreatic islets. However, hydralazine showing the same antihypertensive effect as the highest dose of aliskiren did not exert the same protective effect as aliskiren in db/db mice. Conclusions Our work provides the first evidence that direct renin inhibition protects against cardiovascular and renal complications and pancreatic injury in type 2 diabetes, through the attenuation of oxidative stress.

GW24-e2441 SCN5A mutation with lethal ventricular arrhythmias and invalidation of lidocaine in acute myocardial infarction

Objectives Patients are at high risk for potentially fatal ventricular tachycardia (VT)/ventricular fibrillation (VF) when suffering from acute myocardial infarction (AMI), due to myocardial ischaemia/reperfusion injury or scar formation. Recently, it was reported that SCN5A gene mutations may also contribute to electrical storm complicating AMI. The purpose of this study was to investigate potential SCN5A mutation in patients developing VT/VF during AMI, and reveal underlying cellular electrophysiological mechanism. Methods DNA samples of ten unrelated AMI patients with VT/VF were collected and clinical features were available in medical records. Candidate gene SCN5A was screened by direct sequencing. In expressed mutants, whole cell patch-clamp analysis was used to define the electrophysiologic properties. Current-voltage relationships, peak current, current density, voltage dependence of steady-state activation and inactivation of mutated channels were investigated. Results A missense mutation A1427S located at the S5-S6 extracellular linker of domain III (DIII) in SCN5A gene was identified in a 56-year-old female, which was not observed in 400 healthy control chromosomes of the same ethnic background. In addition, we identified three previously reported SNPs in our subjects, including A29A, H558R and D1818D. Electrophysiological analysis showed obvious reduction of sodium current in A1427S mutant without the shape change of I-V curve. Retrospective analysis of the A1427S carrier’s medical records indicated that lidocaine infusion exacerbated episodes of VT/VF inversely and she finally died of refractory VT/VF complicating AMI. Conclusions We identified a novel SCN5A mutation A1427S that resulted in refractory VT/VF complicating AMI. Loss-of-function of the sodium channel indicates cautiously use of prophylactic lidocaine. Genetic screening could ideally identify the patients at high risk and aid in guiding the optimal therapy.

ASSA13-18-2 Aging Attenuates the Inter-Arm Diastolic Blood Pressure Difference Induced by One Arm Exercise

Objectives It is known that one arm exercise increases the inter-arm diastolic blood pressure difference (dIAD) in young people, but no research was performed in middle-aged and more senior population. This study aimed to determine whether ageing impacts the exercise-induced dIAD in healthy subjects. Methods Normotensive adults (n = 120) were recruited and divided into the young (22.5 ± 1.5y), middle-aged (42.8 ± 4.6y) and senior (61.0 ± 7.0y) groups. The right arm exercise involved performing cycling movements at 60 times/min for three minutes. Bilateral brachial BPs were simultaneously measured with two automatic BP measurement devices before (baseline), immediately (0), 5, 10 and 15 min after the exercise. The difference of bilateral diastolic BPs was calculated as BP l-r and its absolute value of equal or over 10mmHg was recognised as IAD. Results At baseline, the SBP l-r and DBP l-r were similar in three age groups. One arm exercise induced markedly DBP decline in the exercised arm, and then increased DBP l-r and dIAD prevalence in three age groups in an age-dependent manner. The dIAD prevalence increased from baseline of zero to 85% at 0 min in young, 37% in middle-aged and 30% in senior groups. One arm exercise did not significantly change SBP l-r and systolic IAD prevalence in three groups. A reverse correlation was found between the DBPl-r 0 and ages (r = –0.359, P < 0.05), but no correlation between ageing and SBPl-r 0. Conclusions Aging attenuates the levels and duration of inter-arm DBP difference induced by one arm exercise in healthy adults.